Liver tumors if the B6C3F1 mouse, which arose spontaneously or were induced by a single dose of any one of 12 diverse chemical carcinogens at 12 days of age, have been examined for the activation of ras proto-oncogenes. To date, 141 such hepatomas have been screened and ~80% of them contained mutations in codon 61 of the H-ras proto-oncogene; another 5% had activated k-ras genes. The H-ras mutations in these hepatomas have been characterized by selective oligonucleotide hybridization or the by the detection of new restriction sites which result from specific base substitutions in the 61st codon. The distinct patterns of ras mutations observed for each chemical indicated that these alterations are a direct result of the interaction of electrophilic ultimate carcinogenesis with these genes in vivo. These observations are also consistent with ras activation being an early event of hepatocarcinogenesis in B6C3F1 mice. Diethylnitrosamine- induced hepatomas were exceptional since DNAs from 50% of these tumors were unable to morphologically transform NIH 3T3 cells. However, DNA from 3 such hepatomas was capable of conferring the tumorigenic phenotype in nude to an immortal nontumorigenic, Syrian hamster cell line. Attempts to clone these potentially novel non- ras transforming genes by cosmid rescue are in progress. The role of ras activation is also being examined by constructing transgenic C57BL/6N mice with germline activated H-ras genes for crosses with normal C3H/HeN mice. Tumor incidence in the parental line and hybrid offspring with be compared to determine whether the C3H/HeN genetic background is especially susceptible to the phenotypic effects of an activated H-ras gene. Finally, a panel of plasmid probes is being developed which detect restriction fragment length polymorphisms (RELP) between C57BL/6N and C3H/HeN mice. These probes will be used to screen B6C3F1 mouse tumors for losses of heterozygosity in specific chromosomal regions. Analogous studies have implicated tumor suppressor genes in a wide variety of human cancers.